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Telomere Transcripts Target Telomerase in HumanCancer CellsTheresa Kreilmeier 1,2, Doris Mejri 1, Marlene Hauck 3, Miriam Kleiter 2,†
and Klaus Holzmann 1,*1 Institute of Cancer Research, Comprehensive Cancer Center, Medical University of Vienna, Borschkegasse 8a,
Vienna 1090, Austria; [email protected] (T.K.); [email protected] (D.M.)2 Department for Companion Animals and Horses, University of Veterinary Medicine Vienna,
Veterinärplatz 1, Vienna 1210, Austria; [email protected] Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University,
1060 William Moore Drive, Raleigh, NC 27607, USA; [email protected]* Correspondence: [email protected]; Tel.: +43-1-40160-57530† Co-last author.
Academic Editor: Gabriele SaretzkiReceived: 9 June 2016; Accepted: 29 July 2016; Published: 16 August 2016
Abstract: Long non-coding transcripts from telomeres, called telomeric repeat-containing RNA(TERRA), were identified as blocking telomerase activity (TA), a telomere maintenance mechanism(TMM), in tumors. We expressed recombinant TERRA transcripts in tumor cell lines with TAand with alternative lengthening of telomeres (ALT) to study effects on TMM and cell growth.Adeno- and lentivirus constructs (AV and LV) were established for transient and stable expression ofapproximately 130 units of telomere hexanucleotide repeats under control of cytomegalovirus (CMV)and human RNase P RNA H1 (hH1) promoters with and without polyadenylation, respectively.Six human tumor cell lines either using telomerase or ALT were infected and analyzed for TA levels.Pre-infection cells using telomerase had 1%–3% of the TERRA expression levels of ALT cells. AV andLV expression of recombinant TERRA in telomerase positive cells showed a 1.3–2.6 fold increase inTERRA levels, and a decrease in TA of 25%–58%. Dominant-negative or small hairpin RNA (shRNA)viral expression against human telomerase reverse transcriptase (hTERT) results in senescence, notinduced by TERRA expression. Population doubling time, cell viability and TL (telomere length)were not impacted by ectopic TERRA expression. Clonal growth was reduced by TERRA expressionin TA but not ALT cell lines. ALT cells were not affected by treatments applied. Established cellmodels and tools may be used to better understand the role of TERRA in the cell, especially fortargeting telomerase.
Keywords: telomerase; enzyme inhibition; telomere; long non-coding transcript; viral expressionsystems; tumor cell lines; human; canine
1. Introduction
Immortality of tumor cells is a hallmark of cancer and a possible therapeutic target [1].Most tumors activate telomerase as a telomere maintenance mechanism (TMM) to gain infinite growthcapacity [2]. The telomerase reverse transcriptase (TERT) gene encodes the catalytic subunit oftelomerase. Highly recurrent mutations in the TERT promoter were found in over 50 cancer types andare the most common mutations in many cancers, making telomerase activation an attractive target forcancer therapy [3].
Transcripts from telomeres termed telomeric repeat-containing RNA (TERRA or TelRNA)were identified first as developmentally regulated RNA originating from RNA polymerase II [4,5].Telomere transcripts originate in human cells from CpG-island promoters from around half of
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chromosomal ends [6]. TERRA-mimetic oligonucleotides were identified in cell extracts as inhibitorsof telomerase activity (TA) [5]. Furthermore, TERRA transcripts were identified in vitro as naturalligands and direct inhibitors of telomerase [7]. TERRA works mainly via sequestration of telomerase,thus preventing access of the enzyme to the telomere substrate. TERRA is expressed at high levels innon-malignant cells, but at low levels in tumor cells [5,8]. Reduced TERRA expression is associatedwith TA in tumors and cancer cell lines [8,9]. Moreover, in patients with high-grade astrocytomaand detectable TA, elevated TERRA expression may predict a better prognosis [8]. The relationof TERRA with telomere maintenance and cancer is a vital research topic and described in severalreviews [10–15]. TERRA may be a promising biomarker and potential tool in anti-cancer therapy [12,13].Furthermore, such an approach may be independent of species as TA was detected in tumors fromother mammals as well [16].
We hypothesized that the telomeric part of TERRA may work as a potential telomerase-targetingdrug and developed viral systems for transient and stable recombinant expression of telomeric repeatsin human cell cultures for further study of this approach, including in additional species such as canine.
2. Materials and Methods
2.1. Cell Culture
Human tumor cell lines originated from colorectal adenocarcinoma (SW480), from cervicalcarcinoma (HeLa), from glioblastoma multiforme (T98G, YT-BO), and from osteosarcoma (Saos-2,U2OS). YT-BO was established from tumor tissue at the Medical University of Vienna (Vienna, Austria)and the origin of T98G was recently published [8]. Colon tumor cell lines LT97 and Vaco235 were kindlyprovided by Brigitte Marian (Medical University Vienna, Austria) and grown as described [17,18].GM-847 and GM-639 are SV40-immortalized skin fibroblasts, and G-292 osteosarcoma cell lines werekindly provided by Roger Reddel (Children’s Medical Research Institute, Sydney, Australia). All otherhuman tumor cell lines were obtained from the American Type Culture Collection (ATCC, Manassas,VA, USA). Cells were grown at 37 ◦C under 5% CO2 in media with 10% fetal bovine serum (FBS) asrecommended: HeLa in Roswell Park Memorial Institute medium (RPMI)-1640, T98G in MinimumEssential Medium Eagle (MEME) medium with 10µl/ml non-essential amino acids (NEAA) and2 µL/mL pyruvate, Saos-2 and U2OS in McCoy’s. SW480 and YT-BO were grown in RPMI-1640.Canine tumor cell lines originated from soft tissue sarcomas (MBSa, CoFSa, and PSTS), and wereobtained from Marlene Hauck and grown as described [19].
2.2. Adeno- and Lentivirus Constructs
For transient and stable recombinant TERRA expression in adenovirus (AV) and lentivirus (LV)constructs, a 0.8 kbp telomere DNA fragment cloned in pSP73 (Promega, Mannheim, Germany)was kindly provided as pSP73.Sty11 by Yasuhiko Kiyozuka (Kansai Medical University, Osaka,Japan) [20]. The plasmid was established byTitia de Lange (Rockefeller University, New York,NY, USA) [21]. Around 80% of the inserted fragment was successfully validated by sequencingof both strands (VBC Genomics, Vienna, Austria) and contains ≈130 telomeric repeats of bothperfect (TTAGGG) and degenerated (TTGGGG) telomere hexanucleotides. Recombinant AV andLV for transient and stable expression of the telomere fragment were constructed using the Gatewaycloning system (Invitrogen, Lofer, Austria) with polymerase II (CMV) and III (hH1) promoters andrespective termination signals. In brief, pENTR with hH1 promoter and terminator used for smallhairpin RNA (shRNA) expression and pAd/CMV and pAd/PL plasmids were kindly provided byHiroshi Takemori (Osaka University, Japan). Plasmids were ligated by T4 ligase (Fermentas—ThermoFisher Scientific, Vienna, Austria), transformed in TOP10 Chemically Competent Escherichia colistrain (Invitrogen) and grown under kanamycin or ampicillin (Sigma-Aldrich, Vienna, Austria)antibiotic selection pressure. The oligonucleotides for construction of pENTR shRNA were sense5′-GGCCAGTGGAATTCGAGACCAGCTTCAAGAGAGCTGGTCTCGAATTCCACTTTTTTT-3′ and
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antisense 5′-AATTAAAAAAAGTGGAATTCGAGACCAGCTCTCTTGAAGCTGGTCTCGAATTCCACT-3′ targeting eukaryotic translation initiation factor 4A3 (EIF4A3, alternatively termed NMP265 [22])with sequences as published for small interfering RNA (siRNA) [23]. Briefly, single-strandedoligonucleotides were annealed to resulting double-stranded DNA (dsDNA) with sense-loop-antisensestructure and respective overhangs for direct cloning into NotI and EcoRI sites of the pENTR-hH1plasmid. The shRNA structure was removed by restriction with EcoRI (underlined) and after religationto result in pENTR-hH1 with the following recombinant hH1 driven transcript expression cassette:AGTGGAATTCCACTTTTTTT. Bases for transcription start and termination are marked in bold.Next, the EcoRI restriction enzyme cloning site (underlined) located between the hH1 promoterand the terminator of pENTR-hH1 was replaced by insertion cloning of the linker oligonucleotide5′-AATTGTCGAC-3′ with a SalI site (underlined). The inserted telomere fragment was cut out fromthe pSP73 plasmid by restriction enzymes BamHI and BglII (Fermentas) and inserted in sense andantisense orientation into the novel SalI site of pENTR-hH1 after partial fill in of restriction sites byKlenow fragment (Invitrogen). Single bacteria clones with plasmid constructs of telomere fragment insense or antisense orientation were identified by restriction analyses of plasmid DNA and comparisonwith the predicted banding patterns using Clone Manager 9 (Scientific & Educational Software,Cary, NC, USA). From the pENTR-hH1 plasmid with inserted telomere fragment, the H1 promoterwas deleted for use with Gateway expression vectors with built-in expression cassettes using CMVpromoters, like pAD/CMV and pLenti4/V5 (Invitrogen). To this end, the hH1 promoter was cut outwith SacI and BamHI (Fermentas) and the remaining plasmid fragment was treated with Klenowfragment (Invitrogen) to generate blunt ends, ligated and transformed in competent E. coli as describedbefore. All pENTR plasmid constructs were validated by sequencing. The inserted fragments ofthe pENTR plasmids were transferred by Gateway recombination cloning technique (Invitrogen) toadenoviral (pAd/CMV/V5-DEST and pAd/PL-DEST) and lentiviral (pLenti6/BLOCK-iT-DEST andpLenti4/V5-DEST) expression vectors. Recombinant lentivirus was produced by ViraPower LentiviralExpression System as recommended by manufacturer (Invitrogen). AV expressing shRNA againsthuman telomerase reverse transcriptase (hTERT) was constructed from retroviral vector pMKO-1P-C3kindly provided by William C. Hahn (Dana-Farber Cancer Institute, Harvard Medical School, Boston,MA, USA) [24]. In brief, an expression cassette for shRNA with U6 promoter targeting region3114–3134 from the hTERT transcript was cloned into pENTR and transferred into pAd/PL-DEST forAV production as recommended by the manufacturer (Invitrogen). AV expressing dominant-negativehTERT was provided by Silvia Bacchetti (McMaster University, Hamilton, ON, Canada) [25] and AVexpressing enhanced green fluorescent protein (eGFP) was used as control [26]. Recombinant AVswere amplified as described [27]. Virus titers were determined by Adeno-X Rapid Titer Kit (Clontech,Saint-Germain-en-Laye, France) and by eGFP fluorescence-activated cell sorting analysis (FACSCalibur,BD Biosciences Clontech, Singapore). Constant numbers of cultured cells were incubated with varyingnumbers of AV termed multiplicity of infection (MOI) for transient recombinant expression. For stableexpression, cell populations were selected after infection with LV Lenti6 and Lenti4 by growing in thepresence of lethal doses of antibiotics blasticidin and zeocin (Invitrogen), respectively.
From these cell populations, individual cell lines originating from a single clone were establishedby serial dilution assays. Cells were counted by CASY (OMNI Life Science, Raynham, MA, USA),diluted and seeded as one cell per well in 96-well microtiter plates. Assays were performed twice toensure single clonal origin of established cell lines.
2.3. Relative Telomere Length and Expression
DNA and RNA were isolated and relative telomere length (TL) as telomeric content andendogenous TERRA expression were determined by quantitative PCR (qPCR) as described [8]. Relativequantity (RQ) values were calculated from two independent experiments. For recombinant TERRAexpression the following primers were used: exo-5′_f (5′-CGATCCCCGGGTACCGAG-3′), exo-5′_r(5′-CCCCAACCCCAACCGGAAT-3′), exo-3′_f (5′-TTAGGGTTAGGGTTCGGAAT-3′), exo-3′_r
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(5′-AAGTGGAATTGTCGATCTGATA-3′), T7mod (5′-TAATACGACTCACTATAGGGAGAC-3′).Reference 36B4 gene primers for human and canine are described [19].
Telomeric RNA fluorescence in situ hybridization (FISH) was performed as described [4], exceptthat a peanut agglutinin-fluorescein isothiocyanate (PNA-FITC)-(CCCTAA)4 probe (Dako, Glostrup,Denmark) dissolved in a hybridization buffer (70% formamide, 2 µg/µL bovine serum albumin(BSA), 10% dextran sulphate in 2× saline sodium citrate) was used for visualization of TERRA(UUAGGG)-repeats. Slides were dehydrated and mounted in Vectashield mounting medium (VectorLaboratories, Burlingame, CA, USA) with 0.5 µg/mL 4′,6-diamidino-2-phenylindole (DAPI) forimmunofluorescence microscopy as described previously [28].
2.4. Telomerase Activity
Protein extracts for TA analyses were prepared from cells as described [8]. TA was quantifiedas total product generated (TPG) units by qPCR-telomeric repeat amplification protocol (TRAP) [29]with minor modifications. In brief, reactions were setup ice-cooled in 8-µL volume with GoTaq PCRMaster Mix (Promega) including 0.6 µg protein extracts. TA was detected with 200 nM of telomerasesubstrate (TS) and anchored return CX (ACX) primers. RQ-TRAP was performed on ABI PRISM 7500Fast Sequence Detection System (Applied Biosystems, Foster City, CA, USA) under the followingconditions: incubation for 30 min at 30 ◦C, followed by a two-step qPCR with 42 cycles of 30 s at95 ◦C and 30 s at 60 ◦C. Relative TA values were converted into TPG units using a standard curvegenerated by dilution series of telomerase substrate oligonucleotide TSR8 with eight telomeric repeats.Primers and oligonucleotides are described in [8]. One TPG unit corresponds to 0.001 amoles of TSR8extended for 30 min at 30 ◦C. Furthermore, qPCR-TRAP results were validated by polyacrylamide gelelectrophoresis (PAGE)-TRAP [8].
2.5. Cell Growth and Viability
Population doubling level (PDL) and time were calculated based on constant cell numbersseeded with 2 × 105 cells in 6-well plates and counted after seven days by CASY (Omni Life Science)as described [30]. Cell viability rates were determined as recommended using the colorimetric3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Easy4U, Biomedica,Vienna, Austria) as described [19]. In brief, aliquots of 2 × 103 cells were seeded into 96-well plates in100 µL cell line specific growth medium. Cells were screened under the phase-contrast Nikon invertedmicroscope Eclipse TE300 with a TE-FM Epi-Fluorescence attachment (Nikon, Vienna, Austria) 96 hafter seeding and the MTT assay was performed in quadruplicates. Colorimetric data were used tocalculate cell viability rates. Experiments were repeated twice.
2.6. Clonogenicity Assay
Cells were seeded in 6-well plates with 3 mL cell line specific growth medium at a density of100 cells per well. Unattached cells were removed 24 h later and the cultures were then left to grow forseven days. The number of macroscopic visible colonies was assessed after staining with crystal violetas described [31]. Experiments were repeated for a total of three times.
2.7. Senescence-Associated β-Galactosidase Activity
Similar as for PDL, 2 × 105 cells were seeded in 6-well plates with 3 mL cell line specific growthmedium, but treated with AV constructs before the senescence-associated β-galactosidase assay wasperformed as described [32]. In brief, cells were washed with phosphate-buffered saline (PBS), fixedwith formaldehyde/glutaraldehyde buffer, washed and incubated at 37 ◦C with acidic pH bufferedstaining solution for 4 to 6 h. Blue color development was assessed by phase-contrast microscopy.Representative sections with stained and non-stained cells were counted and percentage of blue stainedcells was calculated.
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2.8. Statistical Analysis
Computations were performed with GraphPad Prism version 5 software (San Diego, CA, USA).Mann-Whitney U test was used for RQ group comparisons. A p value≤ 0.05 was considered significant.
3. Results
3.1. TERRA Expression in Human Cell Models
Tumor cell lines with TA or ALT were analyzed for TERRA expression by qPCR (Figure 1).All tumor cells with TA showed less TERRA expression compared to tumor cells without TA. In detail,TERRA RQ levels of the TA positive cell lines T98G, HeLa and SW480 were 1.8%, 1.0%, and 2.9%,respectively, as compared to TA negative cell line Saos-2 (Figure 1A). These results were furthersupported by the study of additional TA positive (n = 17) and negative (n = 3) cell line models(Figure 1B). TERRA levels in TA positive cell lines as compared to Saos-2 were between 1.0% and 17%.
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Computations were performed with GraphPad Prism version 5 software (San Diego, CA, USA).
Mann‐Whitney U test was used for RQ group comparisons. A p value ≤ 0.05 was considered
significant.
3. Results
3.1. TERRA Expression in Human Cell Models
Tumor cell lines with TA or ALT were analyzed for TERRA expression by qPCR (Figure 1). All
tumor cells with TA showed less TERRA expression compared to tumor cells without TA. In detail,
TERRA RQ levels of the TA positive cell lines T98G, HeLa and SW480 were 1.8%, 1.0%, and 2.9%,
respectively, as compared to TA negative cell line Saos‐2 (Figure 1A). These results were further
supported by the study of additional TA positive (n = 17) and negative (n = 3) cell line models (Figure
1B). TERRA levels in TA positive cell lines as compared to Saos‐2 were between 1.0% and 17%.
Figure 1. Telomeric repeat‐containing RNA (TERRA) transcript expression levels in tumor cell
models. (A) Bars depict mean and error bars represent standard deviation (SD) of TERRA relative
quantity (RQ) values as determined by quantitative PCR (qPCR). RQs are normalized to 36B4 as
reference gene and Saos‐2; (B) Scatter blot of cell lines positive for telomerase activity (TA) (n = 6 from
colon tumors: SW620, LT97, HT29, HCT116, Vaco, Caco; n = 11 from brain tumors: CRL‐1718, CRL‐
2020, KG‐MH, KM‐YH, LN‐140, MGC, MR1, U‐373MG, YU‐PM, HTB138, HTB‐186) and alternative
lengthening of telomeres (ALT) (GM‐639, GM‐847, G‐292). RQ value of Saos‐2 is indicated by dashed
line. Lines depict median and interquartile range.
3.2. Transient and Stable Expression of Recombinant TERRA in Human Cell Lines
We infected human tumor cell lines with recombinant AV and LV constructs for expression of
ectopic TERRA transcripts with and without polyadenylation (Figure 2). Primers from the 5′ and 3′
multicloning site regions specific for exogenous TERRA were used to quantify recombinant TERRA
by qPCR (Figure 2A). After infection with recombinant AV constructs, the relative quantity values of
exogenous TERRA transcript levels were determined in relation to 36B4 as reference gene. Transcript
levels of TERRA expressed from CMV and hH1 promoters were highly abundant and similar to
transcript levels of the ribosomal reference gene (Figure 2B). Results of low variability between 5′‐
Figure 1. Telomeric repeat-containing RNA (TERRA) transcript expression levels in tumor cell models.(A) Bars depict mean and error bars represent standard deviation (SD) of TERRA relative quantity(RQ) values as determined by quantitative PCR (qPCR). RQs are normalized to 36B4 as reference geneand Saos-2; (B) Scatter blot of cell lines positive for telomerase activity (TA) (n = 6 from colon tumors:SW620, LT97, HT29, HCT116, Vaco, Caco; n = 11 from brain tumors: CRL-1718, CRL-2020, KG-MH,KM-YH, LN-140, MGC, MR1, U-373MG, YU-PM, HTB138, HTB-186) and alternative lengthening oftelomeres (ALT) (GM-639, GM-847, G-292). RQ value of Saos-2 is indicated by dashed line. Lines depictmedian and interquartile range.
3.2. Transient and Stable Expression of Recombinant TERRA in Human Cell Lines
We infected human tumor cell lines with recombinant AV and LV constructs for expressionof ectopic TERRA transcripts with and without polyadenylation (Figure 2). Primers from the 5′
and 3′ multicloning site regions specific for exogenous TERRA were used to quantify recombinantTERRA by qPCR (Figure 2A). After infection with recombinant AV constructs, the relative quantityvalues of exogenous TERRA transcript levels were determined in relation to 36B4 as reference gene.
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Transcript levels of TERRA expressed from CMV and hH1 promoters were highly abundant and similarto transcript levels of the ribosomal reference gene (Figure 2B). Results of low variability between5′- and 3′-TERRA levels indicate that recombinant TERRA was expressed as full transcript withouttermination. Furthermore, reverse transcription reactions using random hexanucleotide and oligomerdT primers on RNA resulted in similar relative quantities by qPCR of TERRA expressed from CMVconstructs (Doris Mejri and Klaus Holzmann, unpublished work).
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and 3′‐TERRA levels indicate that recombinant TERRA was expressed as full transcript without
termination. Furthermore, reverse transcription reactions using random hexanucleotide and
oligomer dT primers on RNA resulted in similar relative quantities by qPCR of TERRA expressed
from CMV constructs (Doris Mejri and Klaus Holzmann, unpublished work).
Figure 2. Recombinant TERRA expression constructs in human cancer cells. (A) Expression cassettes
of adeno‐ and lentivirus constructs (AV and LV) with specific exo‐5′ and exo‐3′ primer pairs indicated
for detection of recombinant TERRA by qPCR. TERRA expression constructs contain ≈130 telomere
hexanucleotide repeats of TTAGGG and TTGGGG variant sequences under control of human RNase
P RNA H1 RNA polymerase III promoter (hH1) or human cytomegalovirus RNA polymerase II
promoter (CMV). Transcription start point (TSP) and orientation is indicated at hH1 and CMV
promoters marked by green and red boxes, respectively. Polymerase III termination site (term), SV40
polyadenylation signal (pA) and multi‐cloning‐site (mcs) are indicated; (B) Recombinant 5′‐ and 3′‐
TERRA levels in tumor cell lines 48 h after AV infection with multiplicity of infection (MOI) 10.
Recombinant TERRA expression levels from hH1 (AV hH1‐TERRA, upper panel) and CMV (CMV‐
TERRA, lower panel) promoter construct were determined by qPCR in relation to 36B4. Bars depict
mean and error bars depict SD.
Transient expression of recombinant TERRA from AV constructs did not affect cell viability in
the human tumor cell lines studied. Next, cell clones were established by drug selection and two
rounds of serial dilution purification from LV infected cell lines. We analyzed TERRA expression of
AV infected cells and of selected cell clones after LV infection (Figure 3). All tumor cell lines with
expression of recombinant TERRA also showed increased TERRA levels. Short‐time ectopic TERRA
expression from AV with the CMV promoter resulted in a moderate TERRA transcript increase of
27%–35%, in contrast to a strong 105%–155% increase if ectopic TERRA was expressed from the hH1
promoter (Figure 3A). Cell clones with long‐time stable expression of ectopic TERRA from LV
increased TERRA levels by 44%–89% independent of the promoter system used (Figure 3B). LV cell
clones with recombinant TERRA expression showed a moderate increase of signal in the nucleus but
not the cytoplasm by RNA in situ hybridization (Figure 3C). In detail, both ectopic TERRA transcripts
expressed by CMV or hH1 promoters with or without poly(A) tail increased the number of TERRA
foci compared to control in SW480 cells. Additionally, ectopic TERRA transcription with poly(A) tail
compared to transcription without poly(A) tail showed higher staining intensity of the nucleoplasm.
Figure 2. Recombinant TERRA expression constructs in human cancer cells. (A) Expression cassettesof adeno- and lentivirus constructs (AV and LV) with specific exo-5′ and exo-3′ primer pairs indicatedfor detection of recombinant TERRA by qPCR. TERRA expression constructs contain ≈130 telomerehexanucleotide repeats of TTAGGG and TTGGGG variant sequences under control of human RNase PRNA H1 RNA polymerase III promoter (hH1) or human cytomegalovirus RNA polymerase II promoter(CMV). Transcription start point (TSP) and orientation is indicated at hH1 and CMV promoters markedby green and red boxes, respectively. Polymerase III termination site (term), SV40 polyadenylationsignal (pA) and multi-cloning-site (mcs) are indicated; (B) Recombinant 5′- and 3′-TERRA levels intumor cell lines 48 h after AV infection with multiplicity of infection (MOI) 10. Recombinant TERRAexpression levels from hH1 (AV hH1-TERRA, upper panel) and CMV (CMV-TERRA, lower panel)promoter construct were determined by qPCR in relation to 36B4. Bars depict mean and error barsdepict SD.
Transient expression of recombinant TERRA from AV constructs did not affect cell viability in thehuman tumor cell lines studied. Next, cell clones were established by drug selection and two rounds ofserial dilution purification from LV infected cell lines. We analyzed TERRA expression of AV infectedcells and of selected cell clones after LV infection (Figure 3). All tumor cell lines with expression ofrecombinant TERRA also showed increased TERRA levels. Short-time ectopic TERRA expression fromAV with the CMV promoter resulted in a moderate TERRA transcript increase of 27%–35%, in contrastto a strong 105%–155% increase if ectopic TERRA was expressed from the hH1 promoter (Figure 3A).Cell clones with long-time stable expression of ectopic TERRA from LV increased TERRA levels by44%–89% independent of the promoter system used (Figure 3B). LV cell clones with recombinantTERRA expression showed a moderate increase of signal in the nucleus but not the cytoplasm by RNAin situ hybridization (Figure 3C). In detail, both ectopic TERRA transcripts expressed by CMV or hH1promoters with or without poly(A) tail increased the number of TERRA foci compared to control in
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SW480 cells. Additionally, ectopic TERRA transcription with poly(A) tail compared to transcriptionwithout poly(A) tail showed higher staining intensity of the nucleoplasm.Genes 2016, 7, 46 7 of 14
Figure 3. Increase of TERRA levels by recombinant expression. CMV and hH1 TERRA promoter
constructs for expression of polyadenylated and non‐polyadenylated transcripts, respectively. (A,B)
RQ values for TERRA expression were analyzed by qPCR and compared to 36B4 as reference gene
and to enhanced green fluorescent protein (eGFP) infected cells as control; (A) Cells were infected
with AV at MOI 10 and analyzed after 48 h; (B) Cells were infected with LV and single cell clones
were isolated. Representative results in one of three clones per cell line at passage four are shown; (C)
TERRA expression detected by telomeric RNA‐ fluorescence in situ hybridization (FISH) with
fluorescein isothiocyanate (FITC) label in interphase nucleus (4′,6‐diamidino‐2‐phenylindole, DAPI)
of LV clones from SW480 cells at passage four. Cells were treated with 200 U/mL DNase A. Scale bars
of fluorescence micrographs represent 10 μm. Inlays depict a representative nucleus.
3.3. Telomere Length and Cell Growth Behavior
We studied the effects of recombinant TERRA expression on telomeres and on in vitro tumor
cell properties (Figure 4). Infection of tumor cell lines with AV constructs for transient expression of
recombinant TERRA did not trigger cellular senescence in the human tumor cell lines studied.
Recombinant TERRA expression by AV and LV constructs showed effects ±25% on relative TL in
individual cell line models (Figure 4A). However, expression of ectopic TERRA resulted in no general
trend on the telomeric content. LV clones at passage four after serial dilution purification were
analyzed for cell growth, cell viability and clonogenicity as an ability to form in vitro multicellular
colonies (Figure 4B). Cell growth capacity as cumulative population doubling level and cell viability
by MTT assay were not changed by ectopic TERRA expression in all the tumor cell lines studied. In
contrast, clonogenicity was reduced by TERRA expression to 66%–85% exclusively in the tumor cell
models with TA. Thus ectopic TERRA expressed with and without polyadenylation inhibits clonal
growth capacity of tumor cells dependent on TA.
Figure 3. Increase of TERRA levels by recombinant expression. CMV and hH1 TERRA promoterconstructs for expression of polyadenylated and non-polyadenylated transcripts, respectively. (A,B) RQvalues for TERRA expression were analyzed by qPCR and compared to 36B4 as reference gene and toenhanced green fluorescent protein (eGFP) infected cells as control; (A) Cells were infected with AV atMOI 10 and analyzed after 48 h; (B) Cells were infected with LV and single cell clones were isolated.Representative results in one of three clones per cell line at passage four are shown; (C) TERRAexpression detected by telomeric RNA- fluorescence in situ hybridization (FISH) with fluoresceinisothiocyanate (FITC) label in interphase nucleus (4′,6-diamidino-2-phenylindole, DAPI) of LV clonesfrom SW480 cells at passage four. Cells were treated with 200 U/mL DNase A. Scale bars of fluorescencemicrographs represent 10 µm. Inlays depict a representative nucleus.
3.3. Telomere Length and Cell Growth Behavior
We studied the effects of recombinant TERRA expression on telomeres and on in vitro tumorcell properties (Figure 4). Infection of tumor cell lines with AV constructs for transient expressionof recombinant TERRA did not trigger cellular senescence in the human tumor cell lines studied.Recombinant TERRA expression by AV and LV constructs showed effects ±25% on relative TL inindividual cell line models (Figure 4A). However, expression of ectopic TERRA resulted in no generaltrend on the telomeric content. LV clones at passage four after serial dilution purification were analyzedfor cell growth, cell viability and clonogenicity as an ability to form in vitro multicellular colonies(Figure 4B). Cell growth capacity as cumulative population doubling level and cell viability by MTTassay were not changed by ectopic TERRA expression in all the tumor cell lines studied. In contrast,clonogenicity was reduced by TERRA expression to 66%–85% exclusively in the tumor cell modelswith TA. Thus ectopic TERRA expressed with and without polyadenylation inhibits clonal growthcapacity of tumor cells dependent on TA.
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Figure 4. Telomeres and in vitro cell growth properties of tumor cells with recombinant TERRA
expression. Bars depict mean and error bars depict SD of two independent experiments performed in
triplicates. (A) Senescence‐associated (SA) β‐galactosidase (βgal) positive cells (upper panel) and
relative TL analyzed for telomeric content by qPCR as telomere‐specific/single‐copy gene (T/S)
quantity in cells infected with AV at MOI 10 after 48 h (middle panel). Relative TL was analyzed in
single LV cell clones at passage four (lower panel). Results for TL were normalized on Saos‐2 infected
with eGFP (middle panel) and on LV cell clones expressing eGFP (lower panel); (B) LV cell clones at
passage four were simultaneously grown for six passages in culture and cumulative population
doubling levels (PDLs) are shown (upper panel). Cell viability and colony formation values were
normalized on LV cell clones expressing eGFP marked by dashed lines (middle and lower panel).
3.4. Telomerase Activity in Cell Models with Recombinant TERRA
Both recombinant TERRAs with and without polyadenylation expressed by CMV and by hH1
promoter resulted in strong inhibition of TA (Figure 5). Ectopic TERRA by LV and AV constructs
decreased telomerase activity in all TA tumor cell models to 15–27 and 19–36 TPG units, respectively
(Figure 5A). Tumor cell models without TA remained TA inactive. Transient TERRA expression from
AV constructs in T98G, HeLa and SW480 cells reduced TA after 72 h compared to controls to 31%–
33%, 25%–30% and 50%–58%, respectively. This finding is very similar to a decrease of TA as
observed with dominant‐negative or shRNA AV constructs against hTERT. However, the TA
inhibitory mechanism by TERRA is different, as transient knock‐down of hTERT transcripts or
blockage of the telomerase holoenzyme by expression of mutated hTERT causes senescence in TA
addicted tumor cell lines (Figure 5B). The established TERRA cell models can be used to decipher the
function of TERRA in human tumor cell lines as well as those from other species. Expression of
mutated hTERT in canine sarcoma cell lines with TA resulted in senescence and in inhibition of TA,
similar as observed in human cervical carcinoma cells (Figure 5B,C). Screening of a small panel of
different canine tumor cell lines (n = 8) with TA demonstrated less than 1% of TERRA expression
levels compared to human tumor cells without TA (Theresa Kreilmeier, Miriam Kleiter and Klaus
Figure 4. Telomeres and in vitro cell growth properties of tumor cells with recombinant TERRAexpression. Bars depict mean and error bars depict SD of two independent experiments performedin triplicates. (A) Senescence-associated (SA) β-galactosidase (βgal) positive cells (upper panel) andrelative TL analyzed for telomeric content by qPCR as telomere-specific/single-copy gene (T/S)quantity in cells infected with AV at MOI 10 after 48 h (middle panel). Relative TL was analyzed insingle LV cell clones at passage four (lower panel). Results for TL were normalized on Saos-2 infectedwith eGFP (middle panel) and on LV cell clones expressing eGFP (lower panel); (B) LV cell clonesat passage four were simultaneously grown for six passages in culture and cumulative populationdoubling levels (PDLs) are shown (upper panel). Cell viability and colony formation values werenormalized on LV cell clones expressing eGFP marked by dashed lines (middle and lower panel).
3.4. Telomerase Activity in Cell Models with Recombinant TERRA
Both recombinant TERRAs with and without polyadenylation expressed by CMV and by hH1promoter resulted in strong inhibition of TA (Figure 5). Ectopic TERRA by LV and AV constructsdecreased telomerase activity in all TA tumor cell models to 15–27 and 19–36 TPG units, respectively(Figure 5A). Tumor cell models without TA remained TA inactive. Transient TERRA expression fromAV constructs in T98G, HeLa and SW480 cells reduced TA after 72 h compared to controls to 31%–33%,25%–30% and 50%–58%, respectively. This finding is very similar to a decrease of TA as observed withdominant-negative or shRNA AV constructs against hTERT. However, the TA inhibitory mechanismby TERRA is different, as transient knock-down of hTERT transcripts or blockage of the telomeraseholoenzyme by expression of mutated hTERT causes senescence in TA addicted tumor cell lines(Figure 5B). The established TERRA cell models can be used to decipher the function of TERRA inhuman tumor cell lines as well as those from other species. Expression of mutated hTERT in caninesarcoma cell lines with TA resulted in senescence and in inhibition of TA, similar as observed inhuman cervical carcinoma cells (Figure 5B,C). Screening of a small panel of different canine tumor
Genes 2016, 7, 46 9 of 14
cell lines (n = 8) with TA demonstrated less than 1% of TERRA expression levels compared to humantumor cells without TA (Theresa Kreilmeier, Miriam Kleiter and Klaus Holzmann, unpublished work).TERRA levels in canine tumor cells were comparable to levels in human cells with TA (Figure 1).Whether ectopic TERRA transcripts expressed in canine cells work like in human cells, tumor cellsremain to be investigated. These preliminary results suggest the application of canine TERRA cellmodels for comparative research between human and canine tumor and cell lines.
Genes 2016, 7, 46 9 of 14
Holzmann, unpublished work). TERRA levels in canine tumor cells were comparable to levels in
human cells with TA (Figure 1). Whether ectopic TERRA transcripts expressed in canine cells work
like in human cells, tumor cells remain to be investigated. These preliminary results suggest the
application of canine TERRA cell models for comparative research between human and canine tumor
and cell lines.
Figure 5. TA in tumor cell lines with recombinant TERRA expression and with TERT‐blocking AVs.
Bars depict mean and error bars depict SD of two independent experiments performed in triplicates.
(A) LV cell clones at passage four (upper panel) and cell lines infected with AV at MOI 10 after 72 h
(middle and lower panels) analyzed for TA in total product generated (TPG) units by telomeric repeat
amplification protocol (TRAP) assay. Dashed lines represent the detection limit; (B) Human (HeLa)
and canine (MBSa, CoFSa, PSTS) tumor cells infected with AV at MOI 10 were analyzed after 72 h for
eGFP expression (green) by fluorescence microscopy and for SA β‐galactosidase activity (blue) by
phase‐contrast microscopy. Scale bars depict 500 μm; (C) Human and canine tumor cells analyzed for
TA by TRAP assay and normalized to AV eGFP as control.
4. Discussion
We developed recombinant viral tools for transient and stable recombinant expression of
telomeric repeats in human cell culture to target telomerase activity. Application to telomerase
positive tumor cell lines from various origins like glioblastoma and cervical and colorectal carcinoma
showed that ectopic expression of the telomeric part from the long non‐coding RNA TERRA partially
blocks telomerase activity.
Telomeres and telomerase play a distinct role in aging and cancer, making them very attractive
for novel cancer therapies [33]. Furthermore, telomerase was recognized recently to be a central
regulator of all of the hallmarks of cancer and thus becomes a strong strategic focus as a therapeutic
target in human cancer [34]. There are few telomerase‐directed therapies, but telomerase targeting in
cancer remains a challenge as anti‐telomerase therapies remain unproven. Effectiveness of such drugs
Figure 5. TA in tumor cell lines with recombinant TERRA expression and with TERT-blocking AVs.Bars depict mean and error bars depict SD of two independent experiments performed in triplicates.(A) LV cell clones at passage four (upper panel) and cell lines infected with AV at MOI 10 after 72 h(middle and lower panels) analyzed for TA in total product generated (TPG) units by telomeric repeatamplification protocol (TRAP) assay. Dashed lines represent the detection limit; (B) Human (HeLa)and canine (MBSa, CoFSa, PSTS) tumor cells infected with AV at MOI 10 were analyzed after 72 hfor eGFP expression (green) by fluorescence microscopy and for SA β-galactosidase activity (blue) byphase-contrast microscopy. Scale bars depict 500 µm; (C) Human and canine tumor cells analyzed forTA by TRAP assay and normalized to AV eGFP as control.
4. Discussion
We developed recombinant viral tools for transient and stable recombinant expression of telomericrepeats in human cell culture to target telomerase activity. Application to telomerase positive tumorcell lines from various origins like glioblastoma and cervical and colorectal carcinoma showed thatectopic expression of the telomeric part from the long non-coding RNA TERRA partially blockstelomerase activity.
Telomeres and telomerase play a distinct role in aging and cancer, making them very attractive fornovel cancer therapies [33]. Furthermore, telomerase was recognized recently to be a central regulator
Genes 2016, 7, 46 10 of 14
of all of the hallmarks of cancer and thus becomes a strong strategic focus as a therapeutic target inhuman cancer [34]. There are few telomerase-directed therapies, but telomerase targeting in cancerremains a challenge as anti-telomerase therapies remain unproven. Effectiveness of such drugs ontumor cell growth theoretically depends on the initial length of telomeres until the telomeres shorten tothe critical length which initiates growth arrest or death. Such drugs will most likely prove useful formaintenance therapy, rather than a first-line therapy, to control the microscopic residual disease. PhaseII clinical trials report toxicities due to effects on some hematopoietic proliferative cells that exhibitregulated telomerase activity [35]. These off-target effects have led to the repurposing of these drugs totreat patients with essential thrombocythemia [36] and myelofibrosis [37]. These studies revealed thatcurrent drugs may be non-specific in their effect as no changes in telomere lengths during treatmentwere observed and the initial telomere lengths did not predict clinical response. New approaches arebased on small-molecule telomerase substrates that induce telomere uncapping and promise fewerside effects, as this strategy may result in telomere dysfunction independent of the initial telomerelength [38]. Our data indicate that TERRA directly targets telomerase in tumor cells without any furtherin vitro effects except decreased clonogenic growth. The mechanism of TERRA action is thus clearlydifferent to other telomerase and tumor cell targeting approaches. The established tumor cell lines withmodulated TERRA expression are not affected in growth capacity and might thus serve as a modelfor screening of drugs in combination with telomerase inhibition. Furthermore, possible negativeconsequences of TERRA upregulation, such as genomic instability and toxicity in the hematologicsystem, can be evaluated.
In our study, we successfully validated telomerase inhibition by expression of the recombinanttelomere part of TERRA in tumor cell lines. Inhibition was independent if ectopic TERRA wasexpressed with or without polyadenylation signals, representing the two known isoforms [4].Endogenous TERRA is largely transcribed by RNA polymerase II [5]. About 7% of TERRA transcriptsare 3′ end polyadenylated like most RNA polymerase II transcripts, with some indications that otherpolymerases like RNA polymerase I and III also transcribe TERRA [4,5]. The 5′ end of TERRA contains7-methylguanosine cap structures which, together with the poly(A) tail, contribute to its stability.Here, we identified that recombinant ≈800 nt TERRA, expressed by CMV promoter specific for RNApolymerase II and polyadenylated by SV40 signals or by hH1 promoter for RNA polymerase IIIwithout polyadenylation, both inhibited telomerase activity.
Recombinant TERRA localize within the nucleus similar as described for endogenous TERRA [4].Furthermore, different numbers of telomere-associated TERRA foci were detected in ALT and TA cellswith high and low TERRA levels, respectively. In detail, 80% to 100% of U2OS cells using ALT asTMM displayed 20 to 40 foci compared to 3 to 7 foci in approximately 30% of HeLa cells using TA.We observed in SW480 cells with TA and low endogenous TERRA levels that ectopic TERRA expressionincreased the TERRA levels and also the number of TERRA foci. Similarly, increase of TERRA foci wasreported if endogenous TERRA became displaced from telomeric chromatin [4]. Whether recombinantTERRA transcripts overexpressed in cells with TA become associated with telomeric chromatin seemsto depend upon polyadenylation. Ectopic expression of TERRA with polyadenylation in SW480 cellsincreased the non-telomere-associated TERRA in the nucleoplasm. Our results correspond with theobservation that only unpolyadenylated TERRA is associated with telomeric chromatin [39].
Moderate 1.3–2.6-fold increase of the TERRA levels by AV and LV tools resulted in telomeraseinhibition in the target cell lines. This increase by recombinant TERRA expression is remarkably weak,as tumor cells with TA compared with ALT demonstrated significantly lower TERRA expression levelsof only a few percent. Our results of higher TERRA levels in ALT compared to TA cell lines are in linewith published data of mammalian and yeast cells [6,9,40–43]. Direct targeting of telomerase in humanand canine tumor cell lines by dominant-negative or shRNA AV against hTERT showed very similarpartial telomerase inhibition rates as ectopic TERRA expression and suggests a related mechanismof action, but this remains to be proven by further studies. Similar inhibition of canine telomeraseenzyme by dominant-negative hTERT is not surprising as the canine TERT protein has one of the
Genes 2016, 7, 46 11 of 14
highest levels of sequence homology to human TERT among mammals [44]. Furthermore, preliminaryexperiments with canine TA tumor cell lines demonstrated similarly low TERRA levels. Dogs havebeen recognized as a model for telomerase and telomeres in cancer research [16,45,46]. As caninetelomere biology more closely resembles that of human than, for example, the widely-used mouse, thedomesticated dog could provide a useful model system for TERRA studies in the future.
Indeed, TERRA-mimetic oligonucleotides and transcripts were identified as competitive anduncompetitive inhibitors for telomeric DNA by directly binding to telomerase to inhibit telomeraseactivity in cis [5,7]. Furthermore, the ectopically expressed telomeric parts of TERRA might function asdecoys to block localization of telomerase enzyme to short telomere ends as recently suggested [47,48].TERRA might also alter expression levels of essential genes important for telomerase activity, likehTERT and telomerase RNA component (TERC or hTR). Dominant-negative or knock-down of hTERTin some tumor cells results in a decrease of telomerase activity and inhibition of cell proliferationand apoptosis [49–51], but failed to block telomerase activity in others [52,53]. We observed largenumbers of senescent cells within the studied cell lines shortly after expression of dominant-negativeor shRNA AV against hTERT, but this was not observed with ectopic TERRA expression, indicating avery different method of telomerase inhibition or inhibition of functions by TERRA beyond telomerase.
Population doubling time, cell viability, and telomere length were not affected by ectopic TERRAexpression in the tumor cell lines studied. Individual LV cell clones were grown for up to 30 PDLwithout any indication of a change in cell viability and telomere length. However, clonogenicgrowth capacity was reduced in TERRA-expressing LV clones from TA but not from ALT celllines. Telomeric DNA and TERRAs self-associate in vitro to form four-stranded structures calledG-quadruplexes (G4) which are non-canonical nucleic acid secondary structures [54,55]. Expression ofG4-forming RNA was recently shown to influence cellular behavior through the regulation of geneexpression [56]. G4-forming sequences including TERRA may regulate the expression of many genesthrough the formation of chromatin loop structures via telomere position effect over long distances [57].
The established tools and cell models might be valuable for pre-clinical proof of principlevalidations targeting telomerase, especially in combination with other therapeutic approaches.Furthermore, the tools and cell models may be applicable to other species such as canine forcomparative research.
Acknowledgments: We thank Yasuhiko Kiyozuka (Kansai Medical University, Osaka, Japan) and Titia de Lange(Rockefeller University, New York, NY, USA) for providing the plasmid with telomere sequence; Hiroshi Takemori(Osaka University, Osaka Prefecture, Japan) for pENTR and AV plasmids; William C. Hahn (Dana-Farber CancerInstitute, Harvard Medical School, Boston, MA, USA) for the shRNA plasmid against hTERT, and Silvia Bacchetti(McMaster University, Hamilton, ON, Canada) for the AV expressing dominant-negative hTERT. We thankWolfgang Mikulits and Michael Grusch for support with LV plasmids, Edda Veith for construction of pENTR-hH1with shRNA NMP265 and EcoRI cloning sites between promoter and terminator, Christian Stern and SandraSampl for valuable help with experiments. Klaus Holzmann was supported by the Medical Scientific Fund of theMayor of Vienna #10091 and by the Herzfelder’sche Familienstiftung.
Author Contributions: Klaus Holzmann conceived and designed the experiments. Theresa Kreilmeier andDoris Mejri performed experiments and analyzed the data together with Klaus Holzmann and Miriam Kleiter.Marlene Hauck contributed canine cell lines and provided editing and feedback on the manuscript.Klaus Holzmann and Miriam Kleiter wrote the paper and all authors read the final text.
Conflicts of Interest: The authors declare no conflict of interest.
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